Growth of solid tumors is directly dependent on neovascularization. By understanding the mechanisms that control the neovascular response, it may be possible to design therapeutic strategies to selectively prevent or halt pathological vascular growth and restrain cancer progression. Despite its general biological significance and pathological relevance, little is known about natural inhibitors of blood vessel formation. For several years, we have been working on the protein thrombospondin-1 (TSP1), a glycoprotein that, among many functions, suppresses neovascularization. We have studied the actions of TSP1 in MMTV-TSP1 transgenic and in TSP1 null mice. The studies confirmed a role for TSP1 in the inhibition of capillary growth, but also clearly revealed the existence of other pathways that regulate angiogenesis under physiological conditions and in cancer. We have conducted a search for additional proteins that contain sequences similar to the anti- angiogenic domain of TSP1, in the hope of identifying novel genes with similar effects on endothelial growth and angiogenesis, and that could act in redundant or overlapping pathways of inhibition. We have identified and characterized two novel human proteins, named METH-1 and METH-2. These proteins have 52 percent similarity at the amino acid level and constitute a new family which we have referred to as metallospondins. In addition to the anti-angiogenic domain of TSP1, metallospondins contain metalloproteinase and disintegrin-like domains. We generated recombinant METH-1 protein and demonstrated anti-angiogenic activity in the chorioallantoic membrane and cornea pocket assays. The mechanism of action of METH-1 appears to involve both the TSP-1/type I repeats and the disintegrin domain. The former inhibits proliferation, while the latter affects endothelial cell migration in vitro. We propose that METH-1 might function as a regulator of vessel growth in tumors. In fact, we have demonstrated that overexpression of METH-1 in HT1080 cells reduces tumor growth in nude mice. We also predict that the TSP/type I repeats and disintegrin domains of METH-1 are functionally important regions in the regulation of vessel morphogenesis. We therefore propose to: (1) Examine the mechanisms by which the type I domains of METH-1 mediate capillary suppression; (2) Test the functional significance of the metalloproteinase and disintegrin motifs on the progression of angiogenesis; and (3) Investigate the effect of METH-1 overexpression on mammary growth and physiological angiogenesis in the tumor mammary gland. We believe that identification and characterization of potent and novel inhibitors of angiogenesis is of significant biological and therapeutic relevance for pharmacological intervention to suppress vascular growth in tumors.